色素上皮衍生因子抑制糖基化终产物介导人肾小球系膜细胞糖基化终产物受体表达的研究

目的 观察色素上皮衍生因子（PEDF）、晚期糖基化终产物受体（RAGE）的表达及重组PEDF对RAGE表达的抑制作用,探讨PEDF与DN的关系及对DN的保护作用. 方法 采用糖化小牛血清白蛋白（AGE-BSA）体外诱导人肾小球系膜细胞（HRMCs）,Western blot及RT-PCR法分别检测RAGE、PEDF蛋白和mRNA表达. 结果 （1） AGE-BSA（100～400 mg/L）呈浓度梯度减少HRMCsPEDF表达（P＜0.01）,升高RAGE表达（P＜0.01）;（2）重组PEDF蛋白（5～40 nmol/L）呈浓度依赖性抑制AGE-BSA介导RAGE蛋白在HRMCs的表达（P＜0.05）. 结论 AGEs通过降低PEDF表达,增加RAGE表达参与DN的发生,PEDF可能通过抑制AGE-RAGE轴对DN发挥保护作用.     

恒定及波动高糖下牛视网膜血管周细胞凋亡的线粒体机制

目的 研究恒定及波动高糖培养对牛视网膜血管周细胞凋亡的影响,并探讨其线粒体调控机制.方法 体外培养接近融合的视网膜血管周细胞在恒定及波动高糖中孵育6 d后,采用电镜观察周细胞超微结构改变;TUNEL法检测周细胞凋亡;激光共聚焦显微镜检测周细胞线粒体跨膜电位的改变;分光光度计检测周细胞胞质细胞色素c的变化;免疫组化和RT-PCR测定Bax、Bel-2基因的表达.结果 (1)恒定及波动高糖卜周细胞呈现出典型的细胞凋亡改,变,恒定高糖作用强于波动高糖组;(2)恒定及波动高糖培养使用细胞线粒体跨膜电位降低,周细胞线粒体跨膜电位与周细胞凋亡率负相关(r=-0.89,P<0.01);(3)恒定及波动高糖促使周细胞线粒体细胞色素c转移到胞质,胞质细胞色素c浓度增加,与细胞凋亡率正相关(P<0.01);(4)恒定及波动高糖能诱导凋亡基因Bax表达增加,抑制Bcl-2的表达,Bax/Bcl-2比值增加.Bax/Bcl-2比值与线粒体跨膜电位负相关,与胞质细胞色素c浓度正相关,与周细胞凋亡率正相关(均P<0.01).结论 恒定及波动高糖均可诱导培养的周细胞线粒体跨膜电位降低,细胞色素c释放,细胞发生凋亡,且恒定高糖作用强于波动高糖;线粒体凋亡途径在周细胞凋亡中起重要作用,凋亡基因Bax、Bcl-2可能参与其调控.     

Pigment epithelium-derived factor inhibits TNF-alpha-induced interleukin-6 expression in endothelial cells by suppressing NADPH oxidase-mediated reactive oxygen species generation

Pigment epithelium-derived factor (PEDF) has recently been shown to be involved in the pathogenesis of proliferative diabetic retinopathy. Atherosclerosis is an inflammatory-fibroproliferative disease as well. Oxidative stress plays a major role in retinopathy and atherosclerosis. Accordingly, we investigated effects of PEDF on reactive oxygen species (ROS) generation, NF-kappaB activation and interleukin (IL)-6 expression in TNF-alpha-exposed HUVEC. TNF-alpha significantly increased intracellular ROS generation, which was completely blocked by PEDF or diphenylene iodonium, an inhibitor of NADPH oxidase. Further, PEDF completely prevented the TNF-alpha-induced increase in NADPH oxidase activity. PEDF or an antioxidant, N-acetylcysteine, significantly inhibited the TNF-alpha-induced NF-kappaB activation. PEDF inhibited TNF-alpha-induced expression of IL-6 at both mRNA and protein levels. Moreover, TNF-alpha downregulated PEDF mRNA levels. Ligand blot analysis revealed that HUVEC possessed a membrane protein with binding affinity for PEDF. The results demonstrated that PEDF inhibited TNF-alpha-induced NF-kappaB activation and subsequent IL-6 overexpression in HUVEC by suppressing NADPH oxidase-mediated ROS generation. Our present study suggests that PEDF may play an important role in the development and progression of atherosclerosis.     

色素上皮衍生因子与糖尿病视网膜病变

色素上皮衍生因子(PEDF)来源于人视网膜色素上皮细胞,是眼内有效的血管生成抑制剂,对保持正常眼组织透明无血管的状态有重要作用。PEDF能对抗多种促血管生成因子,有针对性地抑制病理性血管生成,延缓糖尿病视网膜病变的进展,发挥保护作用。同时,PEDF在眼部浓度降低可增加糖尿病视网膜病变的危险。其作用机制仍在探索中。外源性应用PEDF及进行基因治疗对眼部血管增生性疾病具有显著疗效。因此,深入研究可能会发现PEDF治疗临床血管增生性疾病更广泛的作用。     

Substrates modified by advanced glycation end-products cause dysfunction and death in retinal pericytes by reducing survival signals mediated by platelet-derived growth factor

Aims/hypothesis Premature death of retinal pericytes is a pathophysiological hallmark of diabetic retinopathy. Among the mechanisms proposed for pericyte death is exposure to AGE, which accumulate during diabetes. The current study used an in vitro model, whereby retinal pericytes were exposed to AGE-modified substrate and the mechanisms underlying pericyte death explored.Methods Pericytes were isolated from bovine retinal capillaries and propagated on AGE-modified basement membrane (BM) extract or non-modified native BM. The extent of AGE modification was analysed. Proliferative responses of retinal pericytes propagated on AGE-modified BM were investigated using a 5-bromo-2-deoxy-uridine-based assay. The effect of extrinsically added platelet-derived growth factor (PDGF) isoforms on these proliferative responses was also analysed alongside mRNA expression of the PDGF receptors. Apoptotic death of retinal pericytes grown on AGE-modified BM was investigated using terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling labelling, mitochondrial membrane depolarisation and by morphological assessment. We also measured both the ability of PDGF to reverse Akt dephosphorylation that was mediated by AGE-modified BM, and increased pericyte apoptosis.Results Retinal pericytes exposed to AGE-modified BM showed reduced proliferative responses in comparison to controls (p<0.05–0.01), although this effect was reversed at low-AGE modifications. PDGF mRNA levels were differentially altered by exposure to low and high AGE levels, and AGE-modified BM caused significantly increased apoptosis in retinal pericytes. Pre-treatment of AGE-modified BM with PDGF-AA and -BB reversed the apoptosis (p<0.05–0.001) and restored Akt phosphorylation in retinal pericytes.Conclusions/interpretation Evidence suggests that substrate-derived AGE such as those that occur during diabetes could have a major influence on retinal pericyte survival. During diabetic retinopathy, AGE modification of vascular BM may reduce bioavailability of pro-survival factors for retinal pericytes.Conflict of interest: None of the authors have a conflict of interest.     

Pigment epithelium-derived factor inhibits oxidative stress-induced apoptosis and dysfunction of cultured retinal pericytes

Pigment epithelium-derived factor (PEDF) is a potent inhibitor of angiogenesis in the mammalian eye, suggesting that loss of PEDF is implicated in the pathogenesis of proliferative diabetic retinopathy. However, a role for PEDF in early diabetic retinopathy remains to be elucidated. Since oxidative stress is thought to be involved in pericyte loss and dysfunction, one of the changes characteristic of early diabetic retinopathy, we investigated whether and how PEDF could protect cultured retinal pericyte against oxidative stress injury. High glucose (30 mM) increased intracellular reactive oxygen species (ROS) generation in pericytes, which was completely blocked by PEDF. High glucose or H2O2 was found to induce growth retardation and apoptotic cell death of pericytes. PEDF completely restored these cytopathic effects on pericytes. An increased ratio of bax to bcl-2 mRNA level with subsequent activation of caspase-3 was observed in high-glucose- or H2O2-exposed pericytes, which was also completely prevented by PEDF. PEDF significantly increased glutathione peroxidase (GPx) mRNA levels and activity in pericytes. Further, PEDF was found to completely inhibit high-glucose- or H2O2-induced increase in a mRNA ratio of angiopoietin-2 to angiopoietin-1 and up-regulation of VEGF mRNA levels in pericytes. PEDF mRNA levels themselves were down-regulated in high-glucose- or H2O2-exposed pericytes. These results demonstrate that PEDF protects against high-glucose- or H2O2-induced pericyte apoptosis and dysfunction through its anti-oxidative properties via GPx induction. Our present study suggests that substitution of PEDF proteins might be a promising therapeutic strategy for treatment of patients with early diabetic retinopathy.     

Irbesartan inhibits advanced glycation end product (AGE)-induced up-regulation of vascular cell adhesion molecule-1 (VCAM-1) mRNA levels in glomerular endothelial cells

Renin-angiotensin system (RAS) plays a central role in the development and progression of diabetic nephropathy. There is a growing body of evidence that advanced glycation end products (AGE) and inflammation contribute to diabetic nephropathy as well. However, the pathophysiological crosstalk between the RAS and AGE in inflammatory reactions in glomerular endothelial cells (ECs) remains unknown. In this study, we examined whether and how irbesartan, an angiotensin II type 1 receptor blocker (ARB), inhibited the AGE-induced vascular cell adhesion molecule-1 (VCAM-1) gene expression in cultured human glomerular ECs. Irbesartan or an anti-oxidant N-acetylcysteine inhibited the AGE-induced increase in reactive oxygen species (ROS) generation and subsequently blocked up-regulation of VCAM-1 mRNA levels in glomerular ECs. AGE significantly stimulated angiotensin II production by glomerular ECs. Furthermore, irbesartan completely suppressed up-regulation of VCAM-1 mRNA levels in AGE plus angiotensin II-exposed glomerular ECs. Our present data suggest that there exists a crosstalk between the RAS and AGE in inflammatory reactions in glomerular ECs. Irbesartan may play a protective role against diabetic nephropathy by blocking the deleterious effects of AGE-elicited angiotensin II and ROS.     

持续及间断高糖培养对牛视网膜血管周细胞凋亡的影响

目的探讨不同浓度的持续高糖和间断高糖对牛视网膜血管周细胞（BRPs）增殖、凋亡及细胞周期的影响。方法分别在低糖、持续及间断高糖下培养BRPs7天;应用MTT比色法观察各组BRPs增殖情况;应用TUNEL法检测BRPs凋亡率;应用流式细胞术观察BRPs细胞周期的情况。结果（1）持续及间断高糖均可抑制BRPs增殖,诱导其凋亡,与对照组比较,P〈0.01,其中35mmol/L持续高糖组的吸光度为0.274,凋亡率为48.73%;（2）持续及间断高糖可使BRPsG0/G1期细胞比例增加,S期细胞比例减少,G2/M细胞比例无明显改变;（3）间断高糖组中波动幅度及频度大者对BRPs的作用更明显。结论高糖毒性和高糖波动性共同参与视网膜病变的发生,且高糖绝对水平的作用超过高糖的不稳定性作用。     

波动性及恒定性高糖对牛视网膜毛细血管周细胞氧化应激的影响

目的 对比研究波动性与恒定性高糖对牛视网膜毛细血管周细胞氧化应激的影响.方法 体外培养3代融合的牛视网膜血管周细胞,分别在对照组(5.5 mmol/L)、不同浓度高糖(15 mmol/L、25 mmol/L)、不同幅度的高糖波动(5.5～15 mmol/L、5.5～25 mmol/L)下孵育6 d.硫代巴比妥酸检测培养液丙二醛(MDA)水平;黄嘌呤氧化酶法检测培养液超氧化物歧化酶(SOD)水平.结果 与对照组相比,波动性高糖组与恒定性高糖组MDA含量/SOD活力比值增加(P＜0.05),且与葡萄糖浓度及高糖波动幅度呈正相关(P＜0.01),波动性高糖1组(5.5～15 mmol/L)较恒定性高糖1组(15 mmol/L)MDA含量/SOD活性比值增加(P＜0.05),恒定性高糖2组(25 mmol/L)较波动性高糖2组(5.5～25 mmol/L)MDA含量/SOD活性比值增加(P＜0.05).结论 在一定葡萄糖浓度范围内,波动性高糖较恒定性高糖对细胞的氧化损伤加重,而超过一定葡萄糖浓度,恒定性高糖使氧化损伤更为显著.     

A study of the effects of human blood derivatives and individual growth factors on [3H]thymidine uptake in bovine retinal pericytes and endothelial cells

Pericytes disappear early, selectively and specifically from retinal capillaries in diabetic microangiopathy, but little is known of their growth and turnover in health and disease. We have studied the effects of human blood derivatives and of a panel of individual growth factors on [³H]thymidine incorporation in bovine retinal pericytes and endothelial cells. Human serum and platelet-rich plasma stimulated incorporation of the nucleotide in a dose-dependent manner in both cell types, and did so more potently than platelet-free plasma. Consistent and significant stimulation of DNA synthesis in pericytes was observed with basic fibroblast growth factor (ED₅₀= 1.8×10^–13 mol/l), acidic fibroblast growth factor (7.4× 10^–12 mol/l), insulin-like growth factor 1 (8.6×10^–10 mol/l), insulin (158 U/ml) and endothelin-1 (6.1×10^–10 mol/l). Transforming growth factor ₁ inhibited DNA synthesis (ID₅₀=3.6×10^–10 mol/l) and so did heparin (1.4×10^–6 mol/l) and low molecular weight heparin (2.9×10^–6 mol/l). Retinal endothelial cells were stimulated by basic fibroblast growth factor (3.2×10^–13 mol/l) and acidic fibroblast growth factor (1.3×10^–9 mol/l), and inhibited by transforming growth factor ₁, (1.6×10^–12 mol/l). Neither cell type was stimulated by platelet-derived growth factor (A+B chain heterodimer), epidermal growth factor, growth hormone, or nerve growth factor (7S complex). The characteristics and active concentrations of the above growth factors suggest that none is solely responsible for the pericyte mitogenic activity of platelets, serum or plasma. Some, though, may play a role in the regulation of pericyte turnover through paracrine mechanisms which should be further investigated.     

Pigment epithelium-derived factor mitigates inflammation and oxidative stress in retinal pericytes exposed to oxidized low-density lipoprotein

Oxidized and/or glycated low-density lipoprotein (LDL) may mediate capillary injury in diabetic retinopathy. The mechanisms may involve pro-inflammatory and pro-oxidant effects on retinal capillary pericytes. In this study, these effects, and the protective effects of pigment epithelium-derived factor (PEDF), were defined in a primary human pericyte model. Human retinal pericytes were exposed to 100 microg/ml native LDL (N-LDL) or heavily oxidized glycated LDL (HOG-LDL) with or without PEDF at 10-160 nM for 24 h. To assess pro-inflammatory effects, monocyte chemoattractant protein-1 (MCP-1) secretion was measured by ELISA, and nuclear factor-kappaB (NF-kappaB) activation was detected by immunocytochemistry. Oxidative stress was determined by measuring intracellular reactive oxygen species (ROS), peroxynitrite (ONOO(-)) formation, inducible nitric oxide synthase (iNOS) expression, and nitric oxide (NO) production. The results showed that MCP-1 was significantly increased by HOG-LDL, and the effect was attenuated by PEDF in a dose-dependent manner. PEDF also attenuated the HOG-LDL-induced NF-kappaB activation, suggesting that the inhibitory effect of PEDF on MCP-1 was at least partially through the blockade of NF-kappaB activation. Further studies demonstrated that HOG-LDL, but not N-LDL, significantly increased ONOO(-) formation, NO production, and iNOS expression. These changes were also alleviated by PEDF. Moreover, PEDF significantly ameliorated HOG-LDL-induced ROS generation through up-regulation of superoxide dismutase 1 expression. Taken together, these results demonstrate pro-inflammatory and pro-oxidant effects of HOG-LDL on retinal pericytes, which were effectively ameliorated by PEDF. Suppressing MCP-1 production and thus inhibiting macrophage recruitment may represent a new mechanism for the salutary effect of PEDF in diabetic retinopathy and warrants more studies in future.     

Pigment epithelium-derived factor (PEDF) prevents diabetes- or advanced glycation end products (AGE)-elicited retinal leukostasis

Pigment epithelium-derived factor (PEDF) is the most potent inhibitor of angiogenesis in the mammalian eye, suggesting that PEDF may protect against proliferative diabetic retinopathy. However, a role for PEDF in early diabetic retinopathy remains to be elucidated. Leukocyte adhesion to retinal capillary endothelium (leukostasis) is a critical event in early diabetic retinopathy, whose process is mainly mediated by intercellular adhesion molecule-1 (ICAM-1). We investigated here whether PEDF could prevent diabetes- or advanced glycation end products (AGE)-elicited retinal leukostasis by suppressing ICAM-1 expression. Immunohistochemistry of 8-hydroxydeoxyguanosine (8-OHdG), an oxidative stress marker, showed intense staining in the nuclei of cells in the inner and outer plexiform layers of streptozotocin-induced diabetic rat retinas. Administration of PEDF or pyridoxal phosphate, an AGE inhibitor, decreased retinal levels of 8-OHdG and subsequently suppressed ICAM-1 gene expression and retinal leukostasis in diabetic rats. Further, intravenous administration of AGE to normal rats increased ICAM-1 gene expression and retinal leukostasis, which were blocked by PEDF. PEDF also inhibited the AGE-induced T cell adhesion to microvascular endothelial cells by suppressing ICAM-1 expression. These results demonstrated that PEDF inhibited diabetes- or AGE-elicited retinal leukostasis by suppressing ICAM-1. Our present study suggests that PEDF may play a protective role against early diabetic retinopathy by attenuating the deleterious effect of AGE.     

血红素加氧酶1在高糖和晚期糖基化终末产物诱导的THP-1细胞氧化应激中的作用

目的 探讨血红素加氧酶1(heme oxygenase-1,HO-1)在高糖和晚期糖基化终末产物(AGE)诱导的人单核细胞氧化应激中的作用.方法 根据是否加用15 mmoL/L葡萄糖(高糖)+100μg/ml AGE或HO-1抑制剂锌原卟啉(zinc protoporphyrin,ZnPP),将人单核细胞白m病细胞株THP-1细胞分为正常糖组(5 mmoL/L)、高糖+AGE组、高糖+AGE+ZnPP组、正常糖+ZnPP组,孵育24 h后收集细胞及培养液上清,榆测各组细胞的活性氧簇(ROS)、培养液上清丙二醛和肿瘤坏死因子α(TNF-α)水平以及HO-1 mRNA和蛋白的表达.结果 高糖+AGE组和正常糖+ZnPP组的ROS、丙二醛和TNF-α水平均显著高于正常糖组(均P<0.05).高糖+AGE+ZnPP组的ROS、TNF-α水平显著高于高糖+AGE组(均P<0.05).高糖+AGE+ZnPP组的HO-1 mRNA和蛋白表达水平显著低于高糖+AGE组(0.39+0.02±0.89 vs 0.09±0.384±0.00 vs 0.81±0.02,均P<0.05).结论 ZnPP通过抑制HO-1表达加重高糖和AGE导致的单核细胞氧化应激,HO-1可能在糖尿病氧化应激中起重要作用.     

晚期糖基化终末产物与糖尿病神经病变

非酶糖基化反应是糖尿病神经病变的重要发病机制之一。持久高血糖使体内晚期糖基化终末产物（AGEs）不断积累,沉积于神经组织的AGEs不仅修饰了细胞骨架蛋白、髓鞘蛋白及基质蛋白等,从而直接损害神经结构与功能,而且还通过增强氧化应激反应,或与神经元表面AGEs受体（RAGE）作用进一步导致神经功能紊乱。AGEs还可引起神经微循环障碍。单用或联用AGEs抑制剂、抗氧化剂、可溶性RAGE及抗RAGE IgG在防止、缓解、逆转糖尿病神经病变中显示出巨大的潜力。     

The relationship of glycaemic level to advanced glycation end-product (AGE) accumulation and retinal pathology in the spontaneous diabetic hamster

Summary To assess the relationship between glucose and advanced glycation end products (AGE) and the relationship between AGE and retinal changes in vivo, we studied the time course of retinopathy over 12 months in trypsin digest preparations and measured glycaemia and retinal AGE in spontaneous diabetic hamsters of mild (MD) and severe (SD) phenotypes. Blood glucose levels were elevated in MD (9.44 ± 0.76 mmol/l) and in SD (3 months: 24.3 ± 1.4 mmol/l; 12 months: 31.7 ± 0.8 mmol/l) over non-diabetic controls (NC: 7.15 ± 0.25 mmol/l; p < 0.05 or less vs MD; p < 0.001 vs SD). Similar relations were found for HbA₁. Retinal AGE in mild diabetes was 405 ± 11.3 arbitrary units (AU) (NC 245 ± 7.7; p < 0.01) after 3 months and remained unchanged. A non-linear increase of AGE over time was found in severe hyperglycaemic hamsters (466 ± 21 AU after 3 months and 758 ± 21 AU after 12 months; p < 0.001 vs MD). Pericyte loss in mild diabetes progressed from –26 % after 3 months to –41 % after 12 months (p < 0.001 vs NC). Whereas the initial pericyte loss in severely diabetic hamsters was identical to the mildly diabetic group, a higher degree of pericyte loss occurred after 12 months (–57 %; p < 0.05 vs MD). Endothelial cell numbers remained unaffected by mild hyperglycaemia, but significantly increased over time in severe diabetes reaching 31.7 % above controls after 12 months (p < 0.001 vs NC and MD). Microaneurysms were absent in all retinae examined. Acellular capillary segments were increased in mild diabetes (3.83 ± 0.31 per mm² of retinal area) and severe diabetes (7.83 ± 0.73) over controls (1.0 ± 0.23). These data suggest that a threshold of glycaemia might exist above which AGE removal systems become saturated. Pericyte loss and acellular capillary formation are associated with mild increases in blood glucose and AGE levels while endothelial cell proliferation requires higher glucose and AGE levels. [Diabetologia (1998) 41: 165–170] Received: 31 July 1997 and in revised form: 29 September 1997     

Toxic action of advanced glycation end products on cultured retinal capillary pericytes and endothelial cells: relevance to diabetic retinopathy

Summary The toxic effects of advanced glycation end products (AGEs) on bovine retinal capillary pericytes (BRP) and endothelial cells (BREC) were studied. AGE-modified bovine serum albumin (AGE-BSA) was toxic to BRP. At a concentration of 500 μg/ml it reduced the BRP number to 48 ± 3 % (p < 0.05) of untreated controls, as determined by cell counting with haemocytometer. AGE-BSA was also toxic to bovine aortic endothelial cells (BAEC) reducing cell number to 84 ± 3.1 % of untreated controls. Under similar conditions, low concentrations (62.5 μg/ml) of AGE-BSA were mitogenic to BREC increasing the cell proliferation to 156 ± 11 % (p < 0.05) above that of untreated controls. At a higher dose of 500 μg/ml AGE-BSA decreased the proliferation of BREC to 85 ± 6 % of untreated controls. Immunoblot analysis demonstrated that BRP and BREC express the p60 AGE-receptor. Retinal capillary bed from the human also stained positively for the p60 AGE-receptor. Addition of 0.25 μg/ml of p60 AGE-receptor antibody was able to block the effects of AGE-BSA on BRP and BREC. The level of binding of [¹²⁵I]-labelled AGE-BSA to the cell surface was small but significant among the three cell types. There was also an increase in the internalized pool of radioligand in BRP and BREC but this was very much lower than in BAEC. In all the cell types the internalized pool of [¹²⁵I]-labelled AGE-BSA was much larger than the amount associated with the cell surface. Degradation products were not detected in the media over the 24-h incubation of the cells with [¹²⁵I]AGE-BSA. The binding of [¹²⁵I]-labelled AGE-BSA to the cell surface was prevented by the addition of p60 AGE-receptor. These results suggest that the interaction of AGE-modified proteins with the membrane-bound AGE-receptor may play an important role in the pathogenesis of diabetic retinopathy. [Diabetologia (1997) 40: 156–164] Received: 23 August 1996; and in revised form: 29 October 1996